A single‐center cohort study of patients with hereditary spherocytosis in Central Europe reveals a high frequency of novel disease‐causing genotypes

Hereditary spherocytosis (HS) is the most common red blood cell (RBC) cytoskeleton disorder and the most frequent cause of chronic hemolytic anemia (CHA) in Northern European ancestries. 1 HS is phenotypically and genetically heterogeneous, caused by mutations that alter the expression and/or structure of transmembrane, adapter, or spectrin ‐ based cytoskeletal proteins essential for RBC stability, shape, and function. 1 These defects lead to the formation of spher-oidal, fragile RBCs that are selectively trapped in the spleen, resulting in CHA, jaundice, gallstone formation, and splenomegaly. 2 Clinical HS phenotypes range from subclinical to transfusion ‐ dependent severe CHA. Total or near ‐ total splenectomies are curative for severe HS. 2 HS ‐ causing defects comprise ANK1 (encoding ankyrin ‐ 1), SPTB (encoding β ‐ spectrin), SPTA1 (encoding α ‐ spectrin), SLC4A1 (encoding band 3), and EPB42 (encoding protein 4.2) mutations. HS occurs in all ethnic groups

Palestine region (n = 4), Iran (n = 1), and Chechnya (n = 1) (Supporting Information S1: Tables S1 and S2).Analyses on patients and both parents were performed in 30/35 (86%) families.Details of diagnostics, clinics, genetic analyses, case reports, and summaries on identified genetic variants are provided in the Supporting Information.
Of the 34 identified variants, 19 (56%) were not previously reported.Six variants were described previously by us (Table 1), and two of these were subsequently reported in other populations (Supporting Information S1: Families F2/F14).The remaining nine variants were first published elsewhere (Table 1 and Supporting Information S1: Tables S4-S6).From the 34 index patients with pathogenic (n = 30) or likely pathogenic (n = 4) variants, 22 had AD inherited heterozygous variants (64.5%), five had AD de novo heterozygous variants (14.5%), three had autosomal recessive inheritance (one homozygous SLC4A1, two compound heterozygous SPTA1, 9%), and four had variants with unknown inheritance patterns (12%) (Table 1).In red, are newly described mutations.In black, are mutations previously reported in the literature.The colors of the circles define the type of mutation: orange for frameshifts, pink for nonsense, light green for intronic, dark green for missense, and blue for splicing mutations.
Clinical characteristics for all 69 patients are summarized in Supporting Information S1: Tables S1a-e (index group) and S2a-d (affected family members).Data for grading disease severity before splenectomy were available for all 35 index patients and 21 affected family members.Five of the 56 (9%) patients had severe phenotypes (ANK1, n = 2; SPTB, n = 2; homozygous SLC4A1, n = 1).Moderate phenotypes were observed in 34/56 (61%) patients (ANK1, n = 17; SPTB, n = 10; SLC4A1, n = 3; compound heterozygous SPTA1, n = 3; causative variants not unequivocally identified, n = 1) and mild phenotypes in 17/56 (30%) patients (SLC4A1, n = 10; SPTB, n = 5; ANK1, n = 2).There were milder phenotypes in patients with heterozygous SCL4A1 variants, and eosin 5-maleimide binding was highest compared to the other variant cohorts (Supporting Information S1: Table S3 and Figure S1).Hemoglobin levels were significantly higher in patients with heterozygous SLC4A1 variants compared to patients with ANK1 variants (p-adjusted <0.001) and compound heterozygous SPTA1 variants (p-adjusted <0.05) (Supporting Information S1: Table S3 and Figure S1).Of note, hemoglobin levels were higher in patients with SPTB missense variants compared to SPTB stop gain variants (p-adjusted <0.05) (Supporting Information S1: Figure S19).However, the number of patients was too low to allow the establishment of robust phenotype correlation with different types of variants in HS candidate genes in our cohort (Supporting Information S1: Figures S7/S8 Compared to other studies, we performed all analyses in our hospital and research institute including all available first-degree family members.This allowed us to identify causative variants in 98.5% of patients; and this accuracy is higher than previously reported (e.g., in studies from Western Europe [89%], North America [97%], 5 and East Asia [85%] 8 ).
We found variants in ANK1 as the most common cause of HS (46%), in line with results from North America (49%) 5 and South Asia (53.2%). 7Lower frequencies of causative ANK1 variants were reported in cohorts from Western Europe (27%) 4 and East Asia (29%). 8top-gain and frameshift mutations were found in 75% of our patients with ANK1 variants, similar to results from East 3 and South Asia. 7Interestingly, the intronic variant leading to a frameshift (ANK1, c.1405−9G>A) appears common and was described in different ancestries (Table 1 and Supporting Information S1: Table S4).
Variants in SPTB were identified in 31% of index patients, similar in frequency to North America (33%), 5 but higher than in Western Europeans (20%). 4The highest incidence of causative SPTB variants was found in Asian HS cohorts. 3,7Of note, all patients carrying causative variants in SPTB in our study had Central European ancestry.Similar to results from Asia, 7 truncating variants were found in most of our SPTB patients.In most populations, <20% of causative defects in SPTB are missense variants. 5,7n 14% of our index patients, causative variants in SLC4A1 were identified, as observed in West European (14%) 4 and North American (13%) 5 cohorts.Missense variants were the most prevalent genomic aberrations (60%), corroborating previous findings. 3,4The patient with the most severe phenotype in our cohort carries a homozygous variant in SLC4A1 (Band3 null Vienna ) and we provide an update on the clinical course since the first publication 13 (Supporting Information S1: Family F19 and Figure S10).
A Western European study identified variants in SPTA1 as the most common cause (36%) of HS. 4 Like in other series (South Asia [6.4%], 7 East Asia [9%], 3 North America [5%] 5 ), 6% of our index patients carried causative SPTA1 variants.Interestingly, all carried the low expression α-LEPRA allele in trans to a high impact SPTA1 variant, and have moderate HS phenotypes.
Genotype-phenotype correlations have corroborated that patients who carry heterozygous variants in ANK1 and SPTB (more often high-impact) are phenotypically similar, and patients with heterozygous SLC4A1 variants (more often missense) have milder hematological phenotypes, 4,5,14 with considerable phenotypical variability in the different genetic subcohorts.
Collectively, we provide the first report on the mutational spectrum of HS in a single center in Central Europe.Family analyses, inhouse standardized laboratory, and clinical investigations, coupled with NGS analyses, allowed the identification of the genetic cause with high accuracy and confidence in 98.5% of patients and the discovery of unusual causative variants, like deep intronic, splicing, CNVs, and a homozygous pathogenic variant in SLC4A1.

T A B L E 1 6 |
Description of the variants in HS genes in 34/35 patients of the index group in whom the genetic cause was unequivocally identified.(continued on next page) Central Europe single-centre study reveals novel HS genotypes T A B L E 1 (Continued)

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Siblings with HS phenotypes were tested; they carried the pathogenic variant.m Both parents were tested and none carried the pathogenic variant and none had HS phenotype.n Details are provided in the Supporting Information.I G U R E 1 Two-dimensional protein representation of (A) ANK1, (B) SLC4A1, (C) SPTB, and (D) SPTA1.